s2fieldsofstudy: Biology Chemistry

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Kinetic Characterization of Cerium and Gallium Ions as Inhibitors of Cysteine Cathepsins L, K, and S

Heavy metal ions can disrupt biological functions via multiple molecular mechanisms, including inhibition of enzymes. We investigate the interactions of human papain-like cysteine endopeptidases cathepsins L, K, and S with gallium and cerium ions, which are associated with medical applications. We compare these results with zinc and lead, which are known to inhibit thiol enzymes. We show that Ga3+, Ce3+, and Ce4+ ions inhibit all tested peptidases with inhibition constants in the low micromolar range (between 0.5 µM and 10 µM) which is comparable to Zn2+ ions, whereas inhibition constants of Pb2+ ions are one order of magnitude higher (30 µM to 150 µM). All tested ions are linear specific inhibitors of cathepsin L, but cathepsins K and S are inhibited by Ga3+, Ce3+, and Ce4+ ions via hyperbolic inhibition mechanisms. This indicates a mode of interaction different from that of Zn2+ and Pb2+ ions, which act as linear specific inhibitors of all peptidases. All ions also inhibit the degradation of insoluble elastin, which is a common target of these peptidases in various inflammatory diseases. Our results suggest that these ions and their compounds have the potential to be used as cysteine cathepsin inhibitors in vitro and possibly in vivo.

Introduction
Cysteine cathepsins are important players in lysosomal and extracellular protein turnover in humans and other animals [1,2]. As the name implies, they are peptidases from the cysteine class, specifically the papain-like family. In humans, there are eleven paralogs that differ in their substrate specificity, endo-/exoproteolytic mode of action, and expression patterns. In this work, we investigated cathepsins L, K, and S. All are closely related members of the cathepsin L-like subgroup of papain-like peptidases that act as endopeptidases [3]. Cathepsin L is ubiquitously expressed and has recently received much attention as one of the peptidases involved in the entry of SARS-CoV-2 into the cell [4]. Both knockdown and inhibition of this peptidase can significantly reduce the infectivity of SARS-CoV-2 [5,6]. Cathepsins K and S are expressed in tissue-specific patterns in accordance with their specific functions. Cathepsin K is a central peptidase in bone turnover [7], whereas cathepsin S plays an important role in antigen presentation [8]. Both are also considered potential drug targets in diseases related to their specific biological functions. Cathepsin K is a promising target for the treatment of osteoporosis [9]. Its inhibitor odanacatib (Merck & Co., New York, NY, USA) was evaluated in a phase 3 clinical trial but unfortunately had to be discontinued due to rare but significant side effects [10]. Nevertheless, cathepsin K remains a viable target in osteoporosis [11]. In addition, the inhibitor MIV-711 (Medivir, Huddinge, Sweden) shows promise for the treatment of osteoarthritis [12]. Cathepsin S, on the other hand, is a target for the treatment of inflammatory diseases, such as rheumatoid arthritis and pulmonary fibrosis, and several cathepsin S inhibitors, e.g., petesicatib (Roche, Basel, Switzerland), have shown potential in clinical trials [13,14]. However, as with cathepsin K, no inhibitor has yet been approved for medical use.
Many metal ions are vital to living organisms when present in small amounts [15]. On the other hand, some are also highly cytotoxic [16] and the degree of their toxicity depends on the ligands bound to the metal ion. For example, the toxicity of Cu 2+ has recently been associated with its detrimental effect on the tricarboxylic acid cycle, a central metabolic pathway in aerobic organisms [17]. Several common heavy metal ions, such as Zn 2+ , Fe 3+ , and Cu 2+ are known inhibitors of thiol enzymes due to their direct interaction with catalytic cysteine residues [18]. Zn 2+ , in particular, plays a well-known role in the control of lysosomal cysteine peptidases [19]. Importantly, Zn 2+ has recently been characterized as a potent inhibitor of the SARS-CoV-2 main protease and viral replication [20]; interestingly, we have also recently discovered that zinc pyrithione exhibits potent in vitro inhibitory activity against SARS-CoV-2 papain-like protease as well as human cathepsin L [21]. Other metal ions are considerably less hazardous, and some are now commonly used for medical purposes. Gallium-68 is a widely used isotope for medical imaging [22] and Ga 3+ ions have no significant toxic effects on cell growth and metabolism [23]. Cerium oxide CeO 2 is commonly used in nanocomposite grafts and has the added advantage of scavenging free radicals [24]. Cerium ions can exist as either Ce 3+ or Ce 4+ and shuttling between the two oxidation states is thought to underlie the antioxidant properties of such Ce-containing nanoparticles [25]. Both forms are cytotoxic only at high concentrations (in the range of~10 −4 M) [26][27][28], but CeO 2 nanoparticles have been found to potentially cause DNA damage [29].
To date, no studies have systematically investigated the effects of gallium or cerium ions on thiol enzymes. Here, we investigate the molecular mechanisms by which Ga 3+ , Ce 3+ , and Ce 4+ ions inhibit selected cysteine endopeptidases of the papain-like family (cathepsins K, L, and S) and compare them with Zn 2+ and Pb 2+ ions. We focus in detail on their kinetic modes of action and their effects on the hydrolysis of the protein substrate bovine neck ligament elastin.

Spectroscopic Analysis of Interaction between Ions and Substrate
Pearson's principle of hard-soft acid-base theory (HSAB) is a simple rule for predicting the stability of complexes formed between metal ions and ligands. Undoubtedly, it is a very useful concept, but we must be aware that it also has some limitations (e.g., it is very general and has no direct quantitative scale for Lewis acid-base strength; treatment of ambidentate reactivity; many exceptions occur, etc.) [30][31][32][33]. Of the ions tested, Zn 2+ and Pb 2+ are considered intermediate (borderline) acids, while Ce 3+ , Ce 4+ , and Ga 3+ ions are hard acids [31,34]. Cerium ions have already been shown to form complexes with amino acids and dipeptides [35,36]. Therefore, we first determined whether Ce 3+ , Ce 4+ , and Ga 3+ ions interacted with the synthetic peptide substrates used in the study or otherwise interfered with the kinetic measurements. We measured the UV-Vis absorption spectra of a representative substrate (Z-Phe-Arg-AMC) in the presence and absence of the metal ions studied (specifically, their salts Ga(NO 3 ) 3 , CeCl 3 , and (NH 4 ) 2 Ce(NO 3 ) 6 , respectively) in reaction buffer (Figure 1). The substrate produced a broad peak with a maximum at 325 nm and a shoulder at 295 nm. The spectra of the Ga 3+ and Ce 4+ salts at a concentration of 0.5 mM, which is comparable to the highest concentrations used in the kinetic experiments, produced signals only below 350 nm, while CeCl 3 absorbed much stronger below 400 nm. Therefore, the spectra of CeCl 3 had to be recorded at a significantly lower concentration (0.05 mM) to obtain a signal intensity comparable to the substrate. Since the excitation wavelength used in the kinetic experiments was 370 nm, all progress curves obtained in the presence of CeCl 3 were corrected to account for the inner filter effect [37]. The spectra of the individual salts mixed with the substrate showed no significant shifts in maxima or deviations in intensity from the theoretical sums of the salt and the substrate alone, leading us to conclude that there are no significant interactions or spectral phenomena under these conditions that would interfere with the kinetic measurements of enzyme activity other than correction for the inner filter effect. Absorption spectra of Ce 3+ , Ce 4+ , and Ga 3+ salts in the presence and absence of the synthetic substrate Z-Phe-Arg-AMC. Spectra were recorded in 50 mM sodium acetate buffer pH 5.5 in a quartz cuvette with an optical path of 1 cm. The concentrations of Ce 4+ and Ga 3+ ions in the experiments shown were 0.5 mM and the concentration of Ce 3+ ions was 0.05 mM. The concentration of the substrate was 5 µM.

Kinetic Mechanisms of Action
Metal ions, which according to the HSAB theory are soft or intermediate Lewis acids, are known inhibitors of thiol enzymes interacting with the thiol group of the active site catalytic cysteine residue, which is itself a soft Lewis base [34]. Kinetically, the interaction with the catalytic cysteine residue implies a linear specific (competitive) inhibition mechanism in which the presence of the metal ion prevents the binding of the substrate in the active site. Examples of such ions include Zn 2+ and Pb 2+ ions, both of which are intermediate Lewis acids and are known to interact with cysteine peptidases [18,19]. However, Ce 3+ , Ce 4+ , and Ga 3+ ions, which are the focus of our study, are hard Lewis acids, and therefore, could affect the activity of cysteine peptidases via other kinetic mechanisms. Therefore, we characterized these interactions using appropriate synthetic substrates to measure and compare the activity of cathepsins L, K, and S in the presence and absence of metal ions. As sources of the individual ions, we used their salt forms CeCl3, (NH4)2Ce(NO3)6 and Ga(NO3)3, respectively. Although the counterions in these salts are not identical (Clvs NO3 -) this is of no practical consequence because these anions were never found to affect the activity of the investigated peptidases at such low concentrations (up to 2 mM). Since the reactions were carried out in 0.1 M sodium acetate buffer, their contribution to the total ionic strength of the reaction mixture was also minimal.
Titration curves in the presence of increasing concentrations of ions were performed first and then complemented with specific velocity plots [40] to diagnose the mechanisms of interaction. The kinetic modification parameters of all cathepsin/metal ion pairs characterized in this work are summarized in Table 1. Our results showed that the above assumptions held true overall. Regarding their activity, the tested ions can be distinguished by two different properties. The first subdivision is between linear (full) and hyperbolic (partial) inhibitors; the second is the distinction between ions acting in a 1:1 stoichiometry and those acting via more complex mechanisms that likely involve the binding of more than one metal ion to the peptidase. Mechanisms with a 1:1 stoichiometry were adequately described using the general modifier mechanism [41], while others were fitted using the Certain cerium complexes have been shown to possess hydrolase activity [38,39]. Therefore, we also investigated whether the cerium and gallium salts had detectable hydrolytic activity toward the substrate Z-Phe-Arg-AMC at a metal ion concentration of 0.5 mM, the highest concentration used in the kinetic experiments. No such activity was detected, indicating that the salts themselves do not generate background activity in the kinetic experiments.

Kinetic Mechanisms of Action
Metal ions, which according to the HSAB theory are soft or intermediate Lewis acids, are known inhibitors of thiol enzymes interacting with the thiol group of the active site catalytic cysteine residue, which is itself a soft Lewis base [34]. Kinetically, the interaction with the catalytic cysteine residue implies a linear specific (competitive) inhibition mechanism in which the presence of the metal ion prevents the binding of the substrate in the active site. Examples of such ions include Zn 2+ and Pb 2+ ions, both of which are intermediate Lewis acids and are known to interact with cysteine peptidases [18,19]. However, Ce 3+ , Ce 4+ , and Ga 3+ ions, which are the focus of our study, are hard Lewis acids, and therefore, could affect the activity of cysteine peptidases via other kinetic mechanisms. Therefore, we characterized these interactions using appropriate synthetic substrates to measure and compare the activity of cathepsins L, K, and S in the presence and absence of metal ions. As sources of the individual ions, we used their salt forms CeCl 3 , (NH 4 ) 2 Ce(NO 3 ) 6 and Ga(NO 3 ) 3 , respectively. Although the counterions in these salts are not identical (Clvs. NO 3 -) this is of no practical consequence because these anions were never found to affect the activity of the investigated peptidases at such low concentrations (up to 2 mM). Since the reactions were carried out in 0.1 M sodium acetate buffer, their contribution to the total ionic strength of the reaction mixture was also minimal.
Titration curves in the presence of increasing concentrations of ions were performed first and then complemented with specific velocity plots [40] to diagnose the mechanisms of interaction. The kinetic modification parameters of all cathepsin/metal ion pairs characterized in this work are summarized in Table 1. Our results showed that the above assumptions held true overall. Regarding their activity, the tested ions can be distinguished by two different properties. The first subdivision is between linear (full) and hyperbolic (partial) inhibitors; the second is the distinction between ions acting in a 1:1 stoichiometry and those acting via more complex mechanisms that likely involve the binding of more than one metal ion to the peptidase. Mechanisms with a 1:1 stoichiometry were adequately described using the general modifier mechanism [41], while others were fitted using the four-parameter logistic equation to obtain estimates for their EC 50 values. Interestingly, the effects of each ion also differed between the peptidases, which are all closely related in terms of structure, catalytic properties, and substrate specificity [42]. Most significantly, cathepsin L was inhibited by all ions via a linear specific mechanism, whereas cathepsins K and S were inhibited by Ce 3+ , Ce 4+ , and Ga 3+ ions via hyperbolic mechanisms. As expected, Zn 2+ and Pb 2+ ions acted as linear specific inhibitors binding in a 1:1 stoichiometry ( Figure 2 and Supplementary Figures S1 and S2). Values of the equilibrium dissociation constants (i.e., inhibition constants, a measure of the binding affinity) for the binding of Zn 2+ ions to the target peptidases were in the range of 10 −6 M for all peptidases tested, whereas the binding of Pb 2+ was weaker (between 2.6 × 10 −5 and 1.4 × 10 −4 M). four-parameter logistic equation to obtain estimates for their EC50 values. Interestingly, the effects of each ion also differed between the peptidases, which are all closely related in terms of structure, catalytic properties, and substrate specificity [42]. Most significantly, cathepsin L was inhibited by all ions via a linear specific mechanism, whereas cathepsins K and S were inhibited by Ce 3+ , Ce 4+ , and Ga 3+ ions via hyperbolic mechanisms. As expected, Zn 2+ and Pb 2+ ions acted as linear specific inhibitors binding in a 1:1 stoichiometry (Figure 2 and Supplementary Figures S1 and S2). Values of the equilibrium dissociation constants (i.e., inhibition constants, a measure of the binding affinity) for the binding of Zn 2+ ions to the target peptidases were in the range of 10 −6 M for all peptidases tested, whereas the binding of Pb 2+ was weaker (between 2.6 × 10 −5 and 1.4 × 10 −4 M). Ga 3+ ions acted as linear inhibitors of cathepsin L but as hyperbolic inhibitors of cathepsins K and S, leaving about 20% of residual enzyme activity at saturation (Figure 3). The determined kinetic mechanism in the case of cathepsin L was linear specific inhibition, i.e., it affected enzyme activity in the same way as Zn 2+ and Pb 2+ ions. The kinetic mechanisms for the interaction with cathepsins K and S were diagnosed as hyperbolic mixed inhibition, trending towards activation at high substrate concentrations. Similar mechanisms were previously diagnosed for small molecule modifiers of cathepsins K and S, which presumably act through allosteric mechanisms [43][44][45], suggesting that this is a common mechanism by which these peptidases may be regulated by chemically diverse modifiers. Furthermore, the affinity of Ga 3+ ions for cathepsin L was one order of magnitude higher than for the remaining two (KX values of 6 × 10 −7 M for cathepsin L versus ~4 × 10 −6 M for cathepsins K and S). The observed trend continued with Ce 4+ ions which acted as linear specific inhibitors of cathepsin L and hyperbolic inhibitors of cathepsins K and S Ga 3+ ions acted as linear inhibitors of cathepsin L but as hyperbolic inhibitors of cathepsins K and S, leaving about 20% of residual enzyme activity at saturation (Figure 3). The determined kinetic mechanism in the case of cathepsin L was linear specific inhibition, i.e., it affected enzyme activity in the same way as Zn 2+ and Pb 2+ ions. The kinetic mechanisms for the interaction with cathepsins K and S were diagnosed as hyperbolic mixed inhibition, trending towards activation at high substrate concentrations. Similar mechanisms were previously diagnosed for small molecule modifiers of cathepsins K and S, which presumably act through allosteric mechanisms [43][44][45], suggesting that this is a common mechanism by which these peptidases may be regulated by chemically diverse modifiers. Furthermore, the affinity of Ga 3+ ions for cathepsin L was one order of magnitude higher than for the remaining two (K X values of 6 × 10 −7 M for cathepsin L versus 4 × 10 −6 M for cathepsins K and S). The observed trend continued with Ce 4+ ions which acted as linear specific inhibitors of cathepsin L and hyperbolic inhibitors of cathepsins K and S (Figure 4 and Table 1). The inhibition mechanisms were diagnosed as hyperbolic mixed in the case of cathepsin K and hyperbolic catalytic in the case of cathepsin S. Again, an affinity for cathepsin L was several-fold higher than for cathepsins K and S (K X values of 0.4 × 10 −7 M for cathepsin L versus~1.2 × 10 −6 M for cathepsins K and S).   All the interactions described above could be fitted with mechanisms assuming a 1:1 binding stoichiometry. In contrast, Ce 3+ ions again acted as linear specific inhibitors of cathepsin L, but titrations of cathepsins K and S yielded kinetic profiles that deviated from "classical" models with 1:1 stoichiometry ( Figure 5). These profiles were fitted using the four-parameter logistic equation, where the value of the coefficient h (equivalent to the Hill coefficient) indicates the deviation from regular 1:1 stoichiometry and the value of EC 50 is an estimate of the overall affinity of the interaction ( Table 1). The binding affinity of Ce 3+ ions for all peptidases was significantly lower than that of Ce 4+ ions (K X and EC 50 values of~1 × 10 −5 M, respectively). In addition, the extent of inhibition of cathepsins K and S at saturation was the weakest of all metal ions, resulting in residual enzyme activity of approximately 60% and 70%, respectively. It should be noted that such behavior is neither unprecedented nor unexpected. Similar profiles were obtained, for example, for the inhibition of human leukocyte elastase by glycosaminoglycans, which the authors attributed to several possible binding modes between the two partners [46]. In this case, the structural basis for this behavior remains to be determined. However, given that metal ions can interact with the side chains of various amino acid residues, interaction with multiple sites on the enzymes is a plausible possibility.

Hydrolysis of Bovine Neck Ligament Elastin
Synthetic substrates are much smaller than natural protein substrates, and although they are convenient for characterizing the kinetic properties of enzymes, the results obtained with their use are only an approximation of the proteolytic activity on protein substrates. In our previous work with hyperbolic, presumably allosteric, modifiers of cathepsin K we observed that these small molecules were often significantly less effective when

Hydrolysis of Bovine Neck Ligament Elastin
Synthetic substrates are much smaller than natural protein substrates, and although they are convenient for characterizing the kinetic properties of enzymes, the results obtained with their use are only an approximation of the proteolytic activity on protein substrates. In our previous work with hyperbolic, presumably allosteric, modifiers of cathepsin K we observed that these small molecules were often significantly less effective when protein substrates were used [44]. To complement the results obtained in the previous section and evaluate the efficacy of the tested metal ions in this context, we used bovine neck ligament elastin as a protein substrate. Elastin is an insoluble, structural protein that is often the target of cysteine cathepsins in pathological conditions [47][48][49][50] and we have previously studied the mechanisms by which cathepsins L, K, and S degrade this insoluble material [51]. All metal ions were tested at 0.5 mM concentration, thus assuming saturation of peptidases with the ions based on the kinetic experiments in the previous section. The results in Figure 6 show that cathepsin L was most resistant to inhibition. Its activity was reduced by all metal ions to between 50% and 70% of the uninhibited enzyme. Ga 3+ ions were the most potent inhibitor, but differences between the ions were not significant. In contrast, Zn 2+ ions were the strongest inhibitor of elastin degradation by cathepsins K and S. All ions reduced the elastinolytic activity to between 10% and 40% of the uninhibited enzyme. Thus, the results obtained for cathepsins K and S corresponded well to the residual activities observed in assays with synthetic substrates. The exception was Ce 3+ ions, which were significantly more potent than in assays with synthetic substrates. Elastin degradation by cathepsin K and S has been proposed to involve exosites on the peptidases [52,53]. It is, therefore, possible that the binding of Ce 3+ ions (and others) to these exosites contributes to their inhibitory effect. respectively. The concentrations of the degradation products were measured after their derivatization with fluorescamine, as described previously [51]. The error bars represent the S.E.M. of at least three replicates.

Discussion
The detrimental effects of heavy metal ions on many enzymes are well-known. However, the details of many of these interactions remain poorly understood. We show that selected metal ions, namely Ce 3+ , Ce 4+ , and Ga 3+ , can inhibit human cysteine cathepsin endopeptidases by different kinetic mechanisms than the specific inhibition observed for ions interacting with catalytic cysteines, e.g., Zn 2+ and Pb 2+ [18,19]. Although the structural basis for these observations remains to be elucidated, given their relatively low cytotoxicity, this approach may offer new opportunities for targeting these enzymes in vitro and in vivo, either by the ions alone or, more feasibly, in complexes with synthetic inhibitors of these peptidases. To date, numerous chemically diverse inhibitors of cysteine cathepsins have been investigated that achieved high selectivity for their targets as well as high affinity with KX values in the nanomolar range [54,55]. However, in the absence of clinically approved inhibitors, further efforts are needed to develop alternative targeting strategies. The affinity of the metal ions tested here is much lower than that of the most advanced inhibitors and they are also expected to interact nonspecifically with many other protein targets in vivo, making their use alone not feasible. However, the formation of defined metal complexes with suitable ligands might result in inhibitors with higher af- respectively. The concentrations of the degradation products were measured after their derivatization with fluorescamine, as described previously [51]. The error bars represent the S.E.M. of at least three replicates.

Discussion
The detrimental effects of heavy metal ions on many enzymes are well-known. However, the details of many of these interactions remain poorly understood. We show that selected metal ions, namely Ce 3+ , Ce 4+ , and Ga 3+ , can inhibit human cysteine cathepsin endopeptidases by different kinetic mechanisms than the specific inhibition observed for ions interacting with catalytic cysteines, e.g., Zn 2+ and Pb 2+ [18,19]. Although the structural basis for these observations remains to be elucidated, given their relatively low cytotoxicity, this approach may offer new opportunities for targeting these enzymes in vitro and in vivo, either by the ions alone or, more feasibly, in complexes with synthetic inhibitors of these peptidases. To date, numerous chemically diverse inhibitors of cysteine cathepsins have been investigated that achieved high selectivity for their targets as well as high affinity with K X values in the nanomolar range [54,55]. However, in the absence of clinically approved inhibitors, further efforts are needed to develop alternative targeting strategies. The affinity of the metal ions tested here is much lower than that of the most advanced inhibitors and they are also expected to interact nonspecifically with many other protein targets in vivo, making their use alone not feasible. However, the formation of defined metal complexes with suitable ligands might result in inhibitors with higher affinity and selectivity. Often, metal complexes are not stable under physiological conditions. A famous example of such complexes is the ruthenium complex NAMI-A with anticancer activity [56]. The instability of the complexes can lead to the release of free metal ions from the complexes which can then act synergistically with other synthetic inhibitors. Therefore, it is also important to study the interactions of free metal ions with their potential biological targets, such as cysteine cathepsins.
In addition, these ions provide new opportunities for studying the structural basis for hyperbolic inhibition of papain-like peptidases and the potentially allosteric mechanisms underlying this kinetic behavior. Furthermore, the size of some of these ions allows for the coordination of multiple enzyme molecules to the same ion. This has previously been observed in a crystal structure of cathepsin K (PDB accession code 3C9E), where the binding of a Ca 2+ ion results in a symmetric cathepsin K dimer [57]. In any case, detailed structural characterization will be required to obtain definitive answers.
A peculiar finding from our experiments is that the inhibition mechanisms differ markedly between cathepsin L and the other two. This suggests binding to different sites on the peptidases and/or different responses of the peptidases to the binding of these ions. Cathepsins K and S are more closely related to each other than to cathepsin L [1], but all three have similar substrate specificity [42]. This finding does, however, complement the fact that hyperbolic inhibitors have been described for cathepsins K and S [43][44][45], whereas no such inhibitors are known for cathepsin L. It is possible that cathepsin L is incapable of such modification due to a much more limited activity space, although it has a similar conformational space to cathepsins K and S [58]. Therefore, an allosteric mode of action of Ce 3+ , Ce 4+ , and Ga 3+ ions cannot be excluded and possibly involves interactions of Ce 3+ ions with multiple sites on cathepsins K and S, as discussed in Section 2.2 Cathepsin L also behaved differently from cathepsins K and S in elastinolytic assays ( Figure 6). These differences could be due to their different kinetic profiles of interaction with the metal ions and/or differences in their interactions with elastin. We have previously shown that cathepsin L adheres much less efficiently to the insoluble substrate than cathepsins K and S [51]. In the same work, we also observed that some proteinaceous inhibitors were poorly efficient in preventing elastin degradation [51]. In any case, this observation reiterates the fact that the efficiency of peptidase inhibitors can also depend on the substrate. It should also be noted that much longer incubation times were required for these assays than for the kinetic experiments. Therefore, it is possible that the oxidation states of some metal ions changed during incubation. Cerium ions in particular are known to undergo redox reactions in aqueous solutions [59,60]. Other effects, such as hydration and hydrolysis of metal ions can also affect their interaction with peptidases [61]. However, it can also be expected that such changes occur in vivo, so these assays approximate in vivo processes in this regard as well. Of course, the physiological concentrations of these metals are much lower than the concentrations used in our experiments. Cerium is present in body fluids in sub-nanomolar concentrations [62], which is much lower than the determined binding constants. Therefore, it is unlikely to affect cathepsin activity. Gallium concentrations in blood are in the micromolar range [63] which is comparable to the K X values determined for cathepsins L, K, and S (Table 1). However, most of the gallium was bound to erythrocytes, which limits its availability, and together with the fact that it can interact with many different target proteins, its specific effect on cathepsins is also unlikely. In comparison, the amount of Ga-68 used for radiolabeling is extremely small (a few pmol) [22], and therefore, unlikely to affect cysteine peptidase activity.
Overall, this study shows that metal ions, such as cerium and gallium can affect the activity of cysteine peptidases to varying degrees and by different mechanisms, and it is among the first detailed studies of the kinetic mechanisms of such interactions. Together with the prospect of developing novel metal complexes for use as inhibitors of these peptidases, it identifies cerium and gallium ions as research tools for in vitro studies of the mechanisms of hyperbolic inhibition and will serve as a basis for further exploration of the interactions between peptidases and metal ions.

UV-Vis Spectroscopy
UV-Vis spectra of Ga(NO 3 ) 3 , CeCl 3 , and (NH 4 ) 2 Ce(NO 3 ) 6 were recorded in 50 mM sodium acetate buffer pH 5.5 in a 1 mL quartz cuvette with an optical path of 1 cm at room temperature on a Varian Cary 50 UV-Vis spectrometer at a scan speed setting "slow". The concentrations of Ce 4+ and Ga 3+ were 0.5 mM and the concentration of Ce 3+ was 0.05 mM. The spectra were recorded in the presence and absence of Z-Phe-Arg-AMC (benzyloxycarbonyl-Phe-Arg-7-amido-4methylcoumarin), one of the substrates used in kinetic measurements. The final concentration of the substrate was 5 µM. Background spectra of buffer alone were recorded separately and subtracted automatically from the samples.

Enzyme Kinetics
Kinetic measurements were performed with recombinant human cathepsins at final concentrations in the 0.2 nM to 2 nM range. Reactions were performed in 0.1 M sodium acetate buffer pH 5.5 in single-use acrylic cuvettes (1 cm × 1 cm). Hydrolysis of all synthetic substrates was monitored fluorimetrically at an excitation wavelength λ ex = 370 nm and emission wavelength λ em = 455 nm. The specific substrates used were Z-Leu-Arg-AMC for cathepsin L, Z-Phe-Arg-AMC for cathepsin K, and Z-Val-Val-Arg-AMC for cathepsin S. Reducing agents (e.g., DTT) were omitted from the reaction mixture to prevent complex formation with metal ions. Enzyme stock solutions were kept on ice and regularly tested to assure no loss of activity during each assay.
Initial reaction rates were determined from the slopes of the progress curves and analyzed in GraphPad Prism software version 9.3 (GraphPad Software, La Jolla, CA, USA) using appropriate kinetic models, as described below.

Elastin Degradation Assays
Degradation of insoluble elastin was performed and analyzed according to the protocol in ref. [51]. Briefly, reactions were performed using 200 µL samples, each containing 1 mg of insoluble bovine neck ligament elastin. The enzymes were added to a final concentration of 0.2 µM and the metal ions to a final concentration of 0.5 mM. Samples were incubated at 37 • C for 120 min on an Eppendorf Thermomixer with shaking at 1200 rpm, and the reactions were then stopped by the addition of trichloroacetic acid to a final concentration of 5% (w/V). Samples were cleared by centrifugation and 100 µL of the supernatant was mixed with 1400 µL of 0.2 M sodium borate buffer and 500 µL of a 0.15 mg/mL solution of fluorescamine in acetone. The fluorescence of the samples was measured at an excitation wavelength of λ ex = 390 nm and emission wavelength of λ em = 480 nm. Blank samples without added enzyme were run in parallel and their fluorescence values were subtracted from the samples. Experiments were performed in three replicates of each sample and each experiment was repeated multiple times to assure reproducibility of the results.

Kinetic Models
All experimental data were processed and analyzed with GraphPad Prism 9.3 (Graph-Pad Software, La Jolla, CA, USA). The same software was also used to generate all graphical representations of the data. To characterize the kinetic mechanisms of all metal ions (X) that are bound in a 1:1 stoichiometry, we used the general modifier mechanism [41] shown in Scheme 1. and the reactions were then stopped by the addition of trichloroacetic acid to a final concentration of 5% (w/V). Samples were cleared by centrifugation and 100 µL of the supernatant was mixed with 1400 µL of 0.2 M sodium borate buffer and 500 µL of a 0.15 mg/mL solution of fluorescamine in acetone. The fluorescence of the samples was measured at an excitation wavelength of λex = 390 nm and emission wavelength of λem = 480 nm. Blank samples without added enzyme were run in parallel and their fluorescence values were subtracted from the samples. Experiments were performed in three replicates of each sample and each experiment was repeated multiple times to assure reproducibility of the results.

Kinetic Models
All experimental data were processed and analyzed with GraphPad Prism 9.3 (GraphPad Software, La Jolla, CA, USA). The same software was also used to generate all graphical representations of the data. To characterize the kinetic mechanisms of all metal ions (X) that are bound in a 1:1 stoichiometry, we used the general modifier mechanism [41] shown in Scheme 1.

Scheme 1.
General modifier mechanism for the interaction between enzyme (E), substrate (S) and modifier (X). Scheme 1. General modifier mechanism for the interaction between enzyme (E), substrate (S) and modifier (X).
In this model, the reaction rate in the presence of modifier X is defined as: where v 0 is the reaction rate in the absence of an inhibitor, K X is the equilibrium dissociation constant of complex EX and σ is equal to [S]/K m . Dimensionless coefficients α and β describe the effects of X on substrate affinity (α) and catalytic rate (β) of the enzyme, respectively. Together, both coefficients define the kinetic mechanism of the inhibitor.
To diagnose the mechanism, we used the specific velocity plot [40]. Specific velocity v 0 /v X is defined as: The plot of v 0 /v X against σ/(1 + σ) produces straight lines that always intersect at v 0 /v X = 1. The x-value (σ/(1 + σ)) of the intersection depends on the kinetic mechanism and is used to diagnose the latter. Values of parameters α, β and K X can be determined from replots, obtained by plotting the values of the straight lines extrapolated to σ/(1 + σ) = 0 (a) and σ/(1 + σ) = 1 (b) versus 1/[X] as defined below: The effect of metal ions which could not be described by the above model was analyzed using a modified form of the four-parameter logistic equation adapted to enzyme inhibition: where v ∞ is the residual reaction rate at saturation, EC 50 is the metal ion concentration necessary to achieve a half-maximal effect, i.e., midway between v 0 and v ∞ , and h is a dimensionless coefficient (equivalent to the Hill coefficient) used to identify deviation from 1:1 binding stoichiometry when h = 1. The value of EC 50 is used here as an empirical comparison term for assessing metal ion efficiency towards different enzymes when the kinetic mechanism cannot be sufficiently described by the general modifier mechanism.


Lactucin, a Bitter Sesquiterpene from Cichorium intybus, Inhibits Cancer Cell Proliferation by Downregulating the MAPK and Central Carbon Metabolism Pathway

Lung cancer, especially adenocarcinoma, is the second most occurring and highest fatality-causing cancer worldwide. Many natural anticancer compounds, such as sesquiterpene lactones (SLs), show promising anticancer properties. Herein, we examined Lactucin, an SL from the plant Cichorium intybus, for its cytotoxicity, apoptotic-inducing, cell cycle inhibiting capacity, and associated protein expression. We also constructed a biotinylated Lactucin probe to isolate interacting proteins and identified them. We found that Lactucin stops the proliferation of A549 and H2347 lung adenocarcinoma cell lines while not affecting normal lung cell MRC5. It also significantly inhibits the cell cycle at G0/G1 stage and induces apoptosis. The western blot analysis shows that Lactucin downregulates the MAPK pathway, cyclin, and cyclin-dependent kinases, inhibiting DNA repair while upregulating p53, p21, Bax, PTEN, and downregulation of Bcl-2. An increased p53 in response to DNA damage upregulates p21, Bax, and PTEN. In an activity-based protein profiling (ABPP) analysis of A549 cell’s protein lysate using a biotinylated Lactucin probe, we found that Lactucin binds PGM, PKM, and LDHA PDH, four critical enzymes in central carbon metabolism in cancer cells, limiting cancer cells in its growth; thus, Lactucin inhibits cancer cell proliferation by downregulating the MAPK and the Central Carbon Metabolism pathway.

Introduction
Lung cancers are the second most occurring cancers and the primary cause of cancerrelated death among both men and women worldwide [1]. In 2018, out of 9.6 million cancer-related deaths, 1.76 million were caused by lung cancers [2]. Non-Small Cell Lung Cancer (NSCLC) accounts for 85% of all lung cancers [3], and adenocarcinoma is the principal subtype makeup for 75-85% of lung cancer and related deaths [4].
Rapid metastasis of lung adenocarcinomata [5] are primarily responsible for their late diagnosis, significantly affecting patient survival [6]. Common causes of lung cancers include smoking, air pollution, occupational exposure, and genetics [6]. However, diet also plays a crucial chemoprophylactic role in lung cancers [7][8][9]. It is more evident from the increasing number of novel anticancer compounds discovered in common foods and drinks [10]. Besides rapid metastasis, the aggressive chemotherapeutic regimen often associated with late-stage lung cancer treatment also contributes to poor patient survival [11]. Natural products used as adjuvant therapy have promoted patient survival and quality of life [12,13]. These have created a demand for natural chemopreventive, chemoprotective, and adjuvant compounds in cancer treatment [3,[12][13][14].
Natural products are an abundant source of novel therapeutics [14]. More than 67% of novel anticancer and antitumor drugs are natural compounds or their analog [14,15], and more than 200 compounds and derivatives are at different stages of drug development and trials [15]. Chicory (Cichorium intybus L.) is a widely distributed plant used as medicine, food, drink, and fodder [16]. It has been the subject of many pharmacological studies [16]. Recently, we reviewed the cytotoxic of Chicory [16], where 31 out of 87 C. intybus metabolites possess anticancer, antitumor, and related bioactivities. Lactucin is a phytometabolite from Chicory, phytochemically categorized as bitter Sesquiterpene Lactones (SLs). SLs have been a subject of interest in cancer research for decades, and many have reached clinical trials [15]. As indicated in Ghantous, Gali-Muhtasib, Vuorela, Saliba, and Darwiche [15] reviews, SLs bioactivity is strictly linked to three conserved structural features, e.g., (i) alkylating center reactivity, (ii) side chain and lipophilicity, and (iii) molecular geometry and electronic features. Since many of the structural features that lactucin shares with other SLs have shown antitumor bioactivity against NSCLC [15], Lactucin will likely possess these properties.
Some plants of the Asteraceae family commonly synthesize Lactucin. It is one of the ingredients of lactucarium, a milky white liquid secreted by several lettuce species, e.g., Lactuca serriola, L. saligna, L. viminea, L. glareosa, L. sativa, [17][18][19], etc. In 1970 Chicory root water extract was reported as a light-sensitive, highly potent antimalarial compound. Later Bischoff et al. [20] attributed it to Lactucin, making Chicory the most famous source of Lactucin. Besides antimalarial properties, Lactucin also exerts or potentially exerts anti-inflammatory [21], sedatives [17], anti-adipogenic [22], and anthelmintics [23] effects. Zhang et al. [24] reported Lactucin induces apoptosis and sub-G 1 cell cycle arrest in HL-60 (human leukemia cancer) cells. Ren et al. [25] reported similar effect on KB (human epidermoid carcinoma; IC 50 = 75 µM), and Bel 7402 (human hepatocellular carcinoma; IC 50 = 55 µM) cells. Apart from these reports, the anticancer effect of Lactucin, especially on lung cancer, remains largely unexplored. A structural activity study (SAR) by Ren, et al. [25] revealed the importance of an ester group (γ-butyrolactone) and one exocyclic methylene group for the antitumor activity of Lactucin-like Guanolides. Wang, et al. [22] reported that Lactucin inhibits adipogenesis by downregulating the JAK2/STAT3 signaling pathway and subsequent clonal expansion. However, the cellular target protein of Lactucin and the affected pathway in antitumor activity is still unknown. Herein, we evaluated the anticancer potency of Lactucin using A549 and H2347 lung adenocarcinoma cell lines in vitro to identify interacting proteins and underlying molecular mechanisms.

Lactucin Inhibits Lung Adenocarcinoma Cells Proliferation
The effect of incrementing concentrations of Lactucin on normal lung cells and lung adenocarcinoma cells upon 24 h exposures was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compared to respective control groups, higher Lactucin doses significantly inhibit A549 (IC 50 = 79.87 µM) and H2347 (IC 50 = 68.85 µM) lung adenocarcinoma cells' proliferation in a dose-dependent manner while not affecting MRC-5 cells' proliferation, Figure 1A. However, at different exposure periods (12,24, and 28 h), Lactucin showed significant inhibitory activity on A549 cell proliferation at 24 h and 48 h exposures at higher doses only, Figure 1B. Given that Lactucin can significantly inhibit lung adenocarcinoma cell proliferation at 24 h, exposure at a higher amount without affecting normal lung cells, we used respective IC 50 concentrations of Lactucin for treating A549 and H2347 cells in further cytological experiments. A549 and H2347 cells treated with incrementing doses of Lactucin for 24 h showed a significant dose-dependent increase in the expression of LC3-II (17 kDa) in H2347 cells; and LC3-I (19 kDa) in A549 and H2347 cells, Figure 1C,D.

Effects of Lactucin on Cell Cycle Progression in Lung Adenocarcinoma Cells
Since Lactucin inhibited lung cancer cell proliferation, its cell cycle inhibitory property and related markers were analyzed. Cytometric analysis of DNA Content of 24 h lactucin treated (with respective IC50 concentrations) lung adenocarcinoma cells A549 and H2347 were performed. It revealed that Lactucin inhibits the cell cycle at the G0/G1 phase, Figure 2A. It was further supported by the western blot (WB) analysis of Lactucin-treated (24 h) adenocarcinoma (A549 and H2347) cell's protein, Figure 2B. Both cyclins (cyclin B1 and cyclin D1) and cyclin-dependent protein kinases (CDKs) (CDK2 and CDK4), which regulate the cell cycle through mutual interactions, were dose-dependently downregulated, Figure 2C and 2D. However, p21 and p53, the two CDKs inhibitors we tested, were dose-dependently upregulated. This result suggests Lactucin inhibits the cell cycle of lung adenocarcinoma cells at the G0/G1 phase by upregulating CDKs inhibitor p21 and p53, downregulating cyclins and CDKs expression. Lactucin significantly induces dose-dependent expression of LC3-II in H2347 cells (C,D), detected by WB analysis. Here * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 indicate significantly different from control.

Effects of Lactucin on Cell Cycle Progression in Lung Adenocarcinoma Cells
Since Lactucin inhibited lung cancer cell proliferation, its cell cycle inhibitory property and related markers were analyzed. Cytometric analysis of DNA Content of 24 h lactucin treated (with respective IC 50 concentrations) lung adenocarcinoma cells A549 and H2347 were performed. It revealed that Lactucin inhibits the cell cycle at the G 0 /G 1 phase, Figure 2A. It was further supported by the western blot (WB) analysis of Lactucintreated (24 h) adenocarcinoma (A549 and H2347) cell's protein, Figure 2B. Both cyclins (cyclin B1 and cyclin D1) and cyclin-dependent protein kinases (CDKs) (CDK2 and CDK4), which regulate the cell cycle through mutual interactions, were dose-dependently downregulated, Figure 2C,D. However, p21 and p53, the two CDKs inhibitors we tested, were dose-dependently upregulated. This result suggests Lactucin inhibits the cell cycle of lung adenocarcinoma cells at the G 0 /G 1 phase by upregulating CDKs inhibitor p21 and p53, downregulating cyclins and CDKs expression.

Effects of Lactucin on Apoptosis in Lung Adenocarcinoma Cells
The apoptosis-inducing capacity of Lactucin was measured by flow cytometry analysis of treated A549 and H2347 cells. As shown in Figure 3A, after 24 h incubation with respective IC 50 concentrations of Lactucin, A549 cell's apoptosis increased from 1.93% to 13.42%, and H2347 cell's apoptosis rose from 1.42% to 40.70%. We also observed significant induction of early apoptosis in A549 (from 3.86% to 18.35%) and H2347 (6.07% to 17.36%) cells, Figure 3A. Apoptosis-related indicators such as Bax, Bcl-2 expression; and Caspase-3, and PARP activation, were also compared through WB, Figure 3B. We observed that Lactucin induced a dose-dependent increase in the concentration of both peptides of cleaved Caspase-3, Figure 3C, cleavage of PARP, expression of Bax, and inhibited the expression of Bcl-2, Figure 3D. The upregulation of Bax, downregulation of Bcl-2, and cleavage induction of Caspase-3 and PARP indicate that Lactucin has apoptosis-inducing properties.

Effects of Lactucin on Apoptosis in Lung Adenocarcinoma Cells
The apoptosis-inducing capacity of Lactucin was measured by flow cytometry analysis of treated A549 and H2347 cells. As shown in Figure 3A, after 24 h incubation with respective IC50 concentrations of Lactucin, A549 cell's apoptosis increased from 1.93% to 13.42%, and H2347 cell's apoptosis rose from 1.42% to 40.70%. We also observed significant induction of early apoptosis in A549 (from 3.86% to 18.35%) and H2347 (6.07% to 17.36%) cells, Figure 3A. Apoptosis-related indicators such as Bax, Bcl-2 expression; and Caspase-3, and PARP activation, were also compared through WB, Figure 3B. We observed that Lactucin induced a dose-dependent increase in the concentration of both peptides of cleaved Caspase-3, Figure 3C, cleavage of PARP, expression of Bax, and inhibited . Lactucin significantly downregulates Cyclin B1 and Cyclin D1 (C), CDK4, and CDK2 (D) and downregulates the p21 and p53 (E) expression in both A549 and H2347 cells in a dose-dependent manner. Data are expressed as the means ± standard deviation of triplicate experiments. * p < 0.05, ** p< 0.01, *** p < 0.001, and **** p < 0.0001 were considered significant.

Lactucin Affects the MAPK Signaling Pathway by Downregulating MEK, ERK Phosphorylation
Effects of Lactucin on metabolic markers of lung cancer cells were observed through WB, Figure 4A. Protein lysate from 24 h 80 µM Lactucin-treated A549 cells showed a different level of expression of metabolic markers, Figure 4B. Lactucin dose-dependently increased the expression of PTEN, a tumor suppressor protein that negatively regulates the activation of Akt to p-Akt, Figure 4 [26]. Dose-dependent MEK 1/2 and ERK activation inhibition indicate that Lactucin inhibits A549 cell proliferation by downregulating the MAPK/ERK pathway. We used a biotinylated Lactucin probe to confirm this and isolated Lactucin interaction proteins from A549 cells.

Lactucin Affects the MAPK Signaling Pathway by Downregulating MEK, ERK Phosphorylation
Effects of Lactucin on metabolic markers of lung cancer cells were observed through WB, Figure 4A. Protein lysate from 24 h 80 μM Lactucin-treated A549 cells showed a different level of expression of metabolic markers, Figure 4B. Lactucin dose-dependently increased the expression of PTEN, a tumor suppressor protein that negatively regulates the activation of Akt to p-Akt, Figure 4 [26]. Dose-dependent MEK 1/2 and ERK activation inhibition indicate that Lactucin inhibits A549 cell proliferation by downregulating the MAPK/ERK pathway. We used a biotinylated Lactucin probe to confirm this and isolated Lactucin interaction proteins from A549 cells.

Lactucin Inhibits A549 Cell Proliferation by Downregulating MAPK/ERK and Central Carbon Metabolism Pathways
Natural Lactucin was conjugated with Propargylamine through Michael's addition reaction. Structure of resulting Lactucin-Propargylamine conjugate [(3aR,4S,9aS,9bR)-4hydroxy-9-(hydroxymethyl)-6-methyl-3-((prop-2-yn-1-ylamino)methyl)-3,3a,4,5,9a,9bhexahydroazuleno [4,5-b]furan-2,7-dione] was confirmed using 1 H Nuclear Magnetic Resonance (NMR), Figure 5A. The yield of the probe was estimated at 50% using High Performance Liquid Chromatography (HPLC). Cytotoxicity of the Lactucin probe and Propargylamine showed that the Lactucin probe exerts similar antiproliferation activity on A549 cells as natural Lactucin does both, doses dependent ( Figure 5B) and time-dependent ( Figure 5C) manners. In contrast, Propargylamine does not exert any significant bioactivity. During the Activity-Based Protein Profiling (ABPP) assay, after Co-Immunoprecipitation (Co-IP), isolated protein lysate resolved on the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed five distinct bands in the treatment column after coomassie blue staining ( Figure 6B). After testing these five bands using Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS) and analyzing them using MASCOT against 2019 human proteins. Many probable matches were found. Functional annotation clustering of LC-MS/MS results with the metabolic markers (upregulated and downregulated) data using DAVID Bioinformatics resources showed Lactucin interacts with several key enzymes of the "Central carbon metabolism in cancer" pathway (Kyoto Encyclopedia of Genes and Genomes, KEGG). These interacting proteins were identified as L01 = Phosphoglycerate mutase 1/2/4 (PGM, 70 kDa), L02 = Pyruvate kinase M1/2 (PKM, 58 kDa), L03 = Pyruvate dehydrogenase E1 subunit alpha 1/2 (PDH, 43 kDa), L04 = Lactate dehydrogenase A (LDHA, 37 kDa), but L05 was not identified. At the beginning of this manuscript, we defined cancer and tumor as proliferation disorders. However, they often show increased glucose and glutamine consumption, lactate secretion rate, glycolysis rate, and modified use of metabolic enzyme isoforms [27]. KEGG "Central carbon metabolism in cancer" pathway (Supplementary Material Figure S1 and Figure 6C) shows the difference in carbon metabolism between normal and malignant cells. Lactucin binding to PGM and PKM lower pyruvate synthesis, which binds to LDHA to lower Lactucin synthesis. Lactucin also binds to PDH, limiting the availability of Acetyl CoA entering the Tricarboxylic Acid (TCA) cycle and ultimately reducing lactate production, a hallmark of a metastasized tumor. Reducing PGM, PKM, and LDHA overexpression is attributed to the upregulation of p53 in response to DNA damage. From the KEGG pathway for NSCLC (Supplementary Material Figure S2 and Figure 6C), we see several oncogenes, such as p53, p21, and Bax, were upregulated; Cyclin D1, Akt, MEK, ERK, and CDK4 were downregulated. During the Activity-Based Protein Profiling (ABPP) assay, after Co-Immunoprecipitation (Co-IP), isolated protein lysate resolved on the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed five distinct bands in the treatment column after coomassie blue staining ( Figure 6B). After testing these five bands using Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS) and analyzing them using MASCOT against 2019 human proteins. Many probable matches were found. Func- to lower Lactucin synthesis. Lactucin also binds to PDH, limiting the availability of Acetyl CoA entering the Tricarboxylic Acid (TCA) cycle and ultimately reducing lactate production, a hallmark of a metastasized tumor. Reducing PGM, PKM, and LDHA overexpression is attributed to the upregulation of p53 in response to DNA damage. From the KEGG pathway for NSCLC (Supplementary Material Figure S2 and Figure 6C), we see several oncogenes, such as p53, p21, and Bax, were upregulated; Cyclin D1, Akt, MEK, ERK, and CDK4 were downregulated.

Discussion
In the present experiment, we observed that Lactucin has significant antiproliferative, G 0 /G 1 cell cycle arrest, and apoptosis-inducing properties on A549 and H2347 lung adenocarcinoma, not on MRC-5 normal lung cells. Firstly, Lactucin-induced time and dose-dependent inhibition of A549 and H2347 cells were observed, and then their IC 50 values were determined to be 79.87 µM and 68.85 µM, respectively. According to WHO [28], for L-6 (rat skeletal myoblast) cells, compounds with an IC 50 above 90 µM are not cytotoxic, IC 50 between 2-80 µM is moderately cytotoxic, and IC 50 below 2 µM is cytotoxic. For natural compounds, Ren, et al. [25] described an IC 50 below 100 µM to be cytotoxic and reported Lactucin inhibits KB and Bel 7402 cells with an IC 50 value of 75 µM and 55 µM, respectively. Lima, et al. [29] considered IC 50 of <40 µg/mL and <4 µg/mL for plant extract and pure compounds respectively to be cytotoxic. According to American National Cancer Institute (NCI), except for fibroblast, an IC 50 of ≤30 µg/mL of plant extract is cytotoxic, but they did not specify pure natural compounds. IC 50 values we observed in adenocarcinomas can thus be considered cytotoxic. The LC3-II and LC3-II/LC3-I ratio increase is a classic sign of autophagy [30]. However, this is not always the case due to the lower sensitivity of LC3-I than LC3-II in WB and the higher degradation rate of LC3-II in the presence of lysosomal protease inhibitor [30,31]. We observed a dose-dependent increase of LC3-II in H2347 and LC3-I in A549 and H2347 cells. It was probably due to the concomitant increase in LC3 production and LC3I to LC3II conversion, rapid degradation of LC3-II, or lower detection of LC3-I in WB. In conclusion, LC3 WB results didn't sufficiently indicate that Lactucin directly induces autophagy. LC3-I and LC3-II increase was probably due to autophagosome increase by some other means [30].
We found Lactucin induces G 0 /G 1 cell cycle arrests from cell cycle analysis. Specific cell cycle-related proteins, such as cyclins and CDKs, positively upregulate the cell cycle, whereas CDKs inhibitors stop the Cyclins and CDKs unit assembly [32]. In this experiment, we observed the downregulation of Cyclin B1, Cyclin D1, CDK2, and CDK4, and the upregulation of p21 and p53. Downregulation of Cyclin D1 and upregulation of p53 were associated with G 0 /G 1 cell cycle arrest and apoptosis [32], which coincides with our findings. Lactucin-induced inhibition of cell proliferation was also reported by Zhang, et al. [24] in human HL-60 leukemia cancer cells and by Wang, et al. [22] in mouse 3T3-L1 fibroblast cells. Though in 3T3-L1 G 0 /G 1 cell cycle arrest was reported, [22] in HL-60 cells, Sub-G 1 cell cycle arrest was reported [24].
We also observed an increase in early and late apoptosis in A549 and H2347 cells induced by Lactucin. Lactucin induces apoptosis by upregulating the expression of mitochondrial apoptosis-related proteins, such as c-Caspase, c-PARP, and Bax, while downregulating the expression of Bcl-2. Zhang, et al. [24] reported Lactucin induces apoptosis in HL-60 cells by swelling up mitochondria and endoplasmic reticulum (ER) on the transition electron microscope (TEM). Jang, et al. [33] reported that Lactucin induces ROS-mediated apoptosis in human renal cancer cell Caki-1 by downregulating Bcl-2 expression and CFLARL stability where Bcl-2 downregulation was at a transcriptional level caused by inactivation of the NF-κB pathway. However, we observed concurrent upregulation of PTEN and downregulation of Akt, MEK, and ERK phosphorylation narrowed down the Lactucin-influenced NSCLC pathway to PI3K/Akt and MAPK/ERK. McCubrey, et al. [34] suggested that ERK can activate the NF-κB transcription factor (nuclear factor immunoglobulin κ chain enhancer-B cell) by phosphorylating and activating inhibitor κB kinase (IKK) through an indirect mechanism. We also performed an ABPP assay on A549 protein lysate using a Lactucin-Propargylamine probe. Structural activity relationship (SAR) studies by Ren, et al. [25] revealed that in SL, the position 8 ester group (γ-butyrolactone) and the methylene group at exocyclic position 11 (α), play a significant role in antitumor activities of Lactucin-like guaianolides ( Figure 5A) [16]. α methylene γ lactone, the' enone' or unsaturated carbonyl system (O=C-C=CH 2 ) was also reported to increase the toxicity towards tumor cells [15]. While synthesizing the Lactucin-Propargylamine probe, we avoided the 8-ester group and 11 methylene group positions mentioned earlier. However, adding the alkynyl group to 11 (α), exocyclic methylene didn't hinder its cytotoxicity against A549 cells ( Figure 5B,C). It also suggests that Lactucin may simultaneously exert its activity through different groups. Proteins isolated using this probe were analyzed in LC-MS/MS and combined with WB results for functional enrichment. In the DAVID functional enrichment of western blot and ABPP peptides, four were identified as key enzymes in central carbon metabolism in the cancer cell ( Figure 6C). Overexpressed and modified central carbon metabolism enzymes are essential to support the exponential growth of the metastasized tumor. Regulating their expression will limit the available energy and thus control or seize tumor growth.
In anticancer therapy, oncogenes and tumor suppressors are two likely targets for inhibiting cancer cells [26]. From the KEGG pathway of NSCLEC by Kanehisa Laboratories, we know that in the case of NSCLC like A549 or H2347, we know the potential oncogene and tumor suppressors targets. DAVID enrichment results showed that in the NSCLC, Lactucin downregulates MEK-ERK pathway, resulting in the downregulation of CyclinD1, CDKs, and upregulation of the p53. p53, in turn, upregulates p21 and Bax. It doesn't show interaction with any of the upstream components of NSCLC. When DAVID enrichment for the KEGG pathway "Central Carbon Metabolism in Cancer" was plotted, it showed four Lactucin interacting enzymes besides the western blot findings. Downregulation of MAPK/ERK observed in Figure 6C also lowers the expression of c-Myc, a proto-oncogene involved in cell cycle progression, apoptosis, and cellular transformation. Low c-Myc inhibits uncontrolled DNA synthesis associated with cancer and thus lowers the expression of PGM, PKM, and LDHA, and eventually downregulates carbon consumption needed to sustain malignancy. Lactucin also binds to PDH, reducing its concentration, and decreasing Pyruvate to Acetyl CoA conversion, thus downregulating the TCA cycle and lactate production. Thus, Lactucin inhibits cell proliferation and simultaneously increases cell cycle arrest in lung adenocarcinoma by reducing the MAPK/ERK and lowering carbon metabolism-related enzyme availability.

Materials and Methods
Firstly, we tested the Lactucin for its cytotoxicity, cell cycle inhibiting, and apoptosisinducing properties on lung adenocarcinoma cell lines. Then, its effect on cell cycle, apoptosis, and other related protein expression, was evaluated by WB analysis. Finally, we constructed a biotinylated Lactucin probe to extract Lactucin-interacting proteins, identified them using LC-MS/MS, and elucidated the molecular mechanisms.

Cell Materials
In the present study, we observed the effect of Lactucin (Shanghai Yuanye Biological Technology Co., Ltd., Shanghai, China) on two lung adenocarcinoma cell lines, namely, A549 (Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China), and H2347 (Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China), as well as one normal lung cell line MRC-5 (National Infrastructure of Cell Line Resource, Beijing, China).

Cell Viability Assay
Subconfluent cells (<90%) were aseptically trypsinized and diluted to 5 × 10 4 cell/mL using complete growth media RPMI-1640 (for A549 and H2347) or EMEM (for MRC-5). Cells were then seeded aseptically in 96 welled plates at 1 × 10 4 cells/well (200 µL/well) and incubated for 12 h at 37 • C in a 5% CO 2 incubator before dosing. After 12 h, we exposed the cells to different concentrations of Lactucin for 24 h. All Lactucin concentrations were prepared using complete growth media and contained equal vehicle volume DMSO (v/v). Cell viability was determined using a MTT cell proliferation kit (M1020, Solarbio Biotechnology, Beijing, China) following the manufacturer's instructions. UV absorbance was scanned at 490 nm using a Spark microtitration plate reader (Tecan, Männedorf, Switzerland). Each experiment was repeated three times, the viability of the control group was set to 100%, and cell viability and IC 50 values were calculated.

DNA Content/Cell Cycle Assay
Subconfluent A549 and H2347 cells (<90%) were cultured at 1 × 10 5 cell/mL concentration using complete growth media RPMI-1640 for 12 h at 37 • C in 5% CO 2 . Cells were then treated with respective IC 50 Lactucin solution or equivalent volume DMSO (v/v) solution and incubated for 24 h. After treatment, cells were trypsinized, washed, and fixed for 24 h. Fixed cells were washed and stained using a Propidium Iodide (PI) Flow Cytometry kit (Abcam, Cambridge, UK) following manufacturer instructions. The DNA contents were scanned using a flow cytometer (CytoFLEX, Beckman Coulter Inc., Miami, FL, USA).

Apoptosis Assay
Subconfluent A549 and H2347 cells (<90%) were cultured at 1 × 10 5 cell/mL concentration using complete growth media RPMI-1640 for 12 h at 37 • C in 5% CO 2 . Cells were then treated with respective IC 50 Lactucin solution or equivalent volume DMSO (v/v) solution and incubated for 24 h. After treatment, cells were trypsinized and washed. Following manufacturer instructions, cells were double-stained using the FITC Annexin V Apoptotic kit (BD Pharmingen, San Diego, CA, USA). Samples were filtered (70 µM Nylon cell strainer, Falcon, Corning, Durham, NC, USA) and scanned using a flow cytometer.

Protein Extraction
5 × 10 6 cell/mL untreated [A549 for Co-IP] and treated (Lactucin and DMSO in A549 and H2347 for WB) cells were washed (1× PBS chilled), scraped (cell scraper, Corning, Durham, NC, USA), collected in 1.5 mL centrifuge tubes, and then washed again twice (1× PBS chilled). Between each washing, cells were centrifuged at 600× g for 5 min at 4 • C. After the final centrifuge, 300-500 µL of chilled modified RIPA buffer (1% PMSF + 1% SDS in RIPA) was added to each tube, briefly vortexed and then homogenized using probe sonicator at 30% power of 3 × 5 s with a 10 s gap in between. Homogenized cells were then left to chill on ice for 1 h. After 1 h, cells were centrifuged at 14000× g at 4 • C for 10 min, and intact cells and nuclear materials were separated by pipetting the supernatant into new centrifuge tubes and stored at −20 • C. The protein concentrations were determined using the Bicinchoninic Acid (BCA) protein assay kit (Solarbio Biotechnology, Beijing, China) following manufacturer instructions. UV absorbance was scanned at 562 nm using a microtitration plate reader.

Synthesis of Lactucin Probe
To synthesize the biotinylated Lactucin probe, Lactucin was conjugated with an alkynyl group through Michael's addition reaction, Figure 6A. To a solution of lactucin (5.0 mg, 0.018 mM) in dry pyridine (0.3 mL), Propargylamine (1.5 mg, 0.027 mM) (innochem, Beijing, China) was added and stirred at 0 • C for 36 h under N 2 air. The reaction was monitored by thin-layer chromatography (TLC). The solvent was concentrated and purified through column chromatography (Dichloromethane-CH 3 OH, 15:1) and dried. For structural confirmation, the probe was analyzed using 1 H-NMR at 600 MHz (Oxford NMR AS600, Abingdon, UK) in deuterated DMSO (d-DMSO, Merck KGaA, Darmstadt, Germany), Figure 5A. The yield of the probe was estimated using HPLC.

Cytotoxicity Test and Protein Labelling by Lactucin Probe
Cytotoxicity of the Lactucin probe and Propargylamine was examined by incubating A549 cells with incriminating (0 to 150 µM) concentrations and incubation periods (12,24, and 48 h) of each. The total A549 protein lysate was prepared using the method described in Section 4.6. The Lactucin binding proteins were labeled and pulled out using a biotinylated Lactucin probe following the ABPP method adopted from the method described by Speers and Cravatt [35]. Two 400 µL of protein lysate (2 mg/mL in PBS) were aliquoted into a 1.5mL microcentrifuge tube, and Lactucin-probe or Propargylamine was added (final concentration 100 µM). Both tubes were incubated at RT overnight in a shaker. It was followed by the addition of biotin-azide (Institute of Medicinal Plant Development, Beijing, China) (final concentration 100 µM), then Tris (2-carboxyethyl) phosphine (TCEP, T1656) (final concentration 1 mM), and Tris [(1-benzyl-1H-1,2,3-triazole-4-yl)methyl]-amine (TBTA, T2993) (final concentration 100 µM) (Tokyo Chemical Industry, Japan), and Copper sulfate pentahydrate (CuSO 4 ·5H 2 O) (final concentration 1 mM) (Merck KGaA, Darmstadt, Germany) to each tube with vortex after each addition. Both tubes were incubated at room temperature for 1 h with a vortex every 30 min. Then the tubes were centrifuged at 12,000× g for 10 min at 4 • C, and the supernatant was removed. Protein precipitate was dissolved by adding 750 µL of pre-cooled methanol and sonicating for 3-4 s at 4 • C using a probe sonicator (~30% power level). It was followed by methanol wash thrice with centrifugation (12,000× g, 4 • C for 10 min) in between.

Streptavidin Enrichment
Streptavidin enrichment was by method adopted from the method described by Speers and Cravatt [35]. Probed protein precipitates were dissolved in PBS using 600 µL of 0.2% SDS. Then 100 µL of streptavidin agarose resin (Pierce™ 20347, Thermo Fisher Scientific, Waltham, MA, USA) was added and mixed for 1 h in a shaker at RT. It is followed by washing with 1 mL of 1× PBS with centrifugation (for 1 min at 2500× g) in between and each time collection of supernatants. Finally, the streptavidin beads were eluted by adding 100 µL of 2× SDS-loading buffer, boiling in a water bath for 10 min. Cooling at 4 • C and centrifuging at 2500× g for 1 min. The supernatants were collected and stored at −20 • C until use.

SDS-PAGE and Staining
Protein samples collected from the previous step were resolved in 10% SDS-PAGE gel and visualized by boiling in 0.25% Coomassie Brilliant Blue (CBB) stain for 5 min [26]. The blue-stained gel was washed twice by boiling it with distilled water for 5-10 min. Protein bands from sample columns were cut and stored (at −20 • C) until the identification experiment.

Data Analysis
A bar diagram was plotted for the cytotoxicity test to visualize the "mean percent of inhibition ± standard error" of cell proliferation using Microsoft ® Excel 2019 software. The significance of the lactucin doses was compared with control sample data and calculated by "one-way ANOVA" using "IBM ® SPSS ® Statistics 26". Significant difference at p 0.05, 0.01, 0.001, and 0.0001 was calculated and expressed with different Asterix "*" numbers on the chart or the data table legend. IC 50 was calculated by plotting the Log10 dose on X-axis versus the Normalized response on the Y-axis in Graph Pad Prism 7. Cell cycle cytometry data were analyzed using ModFit LT™ (Version 5.0.9). Apoptosis compensation calculation and data analysis were done using CytExpert™ (Version 2.3.0.84). Protein concentrations were calculated in Microsoft ® Excel 2019. WB bands were quantified using ImageJ ® (Version 1.52a) and analyzed using Graph Pad Prism 7. NMR data was analyzed, the structure was predicted using MestReNova™, and the chemical structure was made using ChemDraw ® Professional. LC-MS/MS results analyzed in MASCOT were combined with WB markers data, and functional enrichment was performed to predict the involved molecular pathway using DAVID Bioinformatics resources [36,37].

Conclusions
In developing anticancer therapy, discovering natural compounds and their mechanisms are the new frontier. They can concurrently affect multiple cell lines, and are helpful in attending to metastasized cancers, while still possessing molecular specificity towards different oncogenes and tumor suppressors. Herein, we reported cytotoxic, cell cycle inhibitory, and apoptosis-inducing properties of Lactucin in A549 and H2347 cells for the first time. We also narrowed down Lactucin's potential target in the A549 cell as MAPK/ERK pathway. Lactucin exerts its effect by lowering the carbon metabolism necessary for metastasized tumor development. To the best of our knowledge, this study was the first attempt at identifying Lactucin's anticancer mechanism in the lung cancer cell line.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27217358/s1, Figure S1: Functional enrichment of ABPP and WB peptides using DAVID Bioinformatics resources predicted Lactucin's interaction with several "Central carbon metabolism in cancer" pathways enzymes (the chart is from KEGG database); Figure S2: Downregulation of the MEK-ERK signaling pathway in NSCLC leads to upregulation of p53 and DNA repair, which downgrade increased carbon consumption-related enzymes (the chart is from KEGG database, red star p < 0.05).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.


Transient Expression of Flavivirus Structural Proteins in Nicotiana benthamiana

Flaviviruses are a threat to public health and can cause major disease outbreaks. Tick-borne encephalitis (TBE) is caused by a flavivirus, and it is one of the most important causes of viral encephalitis in Europe and is on the rise in Sweden. As there is no antiviral treatment available, vaccination remains the best protective measure against TBE. Currently available TBE vaccines are based on formalin-inactivated virus produced in cell culture. These vaccines must be delivered by intramuscular injection, have a burdensome immunization schedule, and may exhibit vaccine failure in certain populations. This project aimed to develop an edible TBE vaccine to trigger a stronger immune response through oral delivery of viral antigens to mucosal surfaces. We demonstrated successful expression and post-translational processing of flavivirus structural proteins which then self-assembled to form virus-like particles in Nicotiana benthamiana. We performed oral toxicity tests in mice using various plant species as potential bioreactors and evaluated the immunogenicity of the resulting edible vaccine candidate. Mice immunized with the edible vaccine candidate did not survive challenge with TBE virus. Interestingly, immunization of female mice with a commercial TBE vaccine can protect their offspring against TBE virus infection.

Introduction
Flaviviruses like yellow fever virus (YFV), dengue virus (DENV), tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV) are a threat to public health and have recently caused several disease outbreaks [1,2]. In particular, TBE has been classified as a notifiable disease in Sweden since July 2004, and the number of TBE cases has increased over the years [3]. According to Sweden's public health agency, a record high number of TBE cases (n = 533) were reported in 2021 [4]. As there is no antiviral treatment available, vaccination remains the best protective measure against TBE. However, current TBE vaccines have limitations as they must be delivered by intramuscular injection, have a burdensome immunization schedule, and may fail due to poor immunogenicity in certain populations [5]. One promising strategy that could help improve flavivirus vaccines in general, and TBE vaccines in particular, is molecular farming, which is the production of pharmaceutically important recombinant proteins in plants. Molecular farming has several advantages over other eukaryotic protein production systems, including simplicity, speed, scalability, safety, and sustainability [6][7][8]. Compared to molecular farming, classical protein production in prokaryotic or other eukaryotic cells is expensive, laborious, and risks contamination with endotoxins or pathogens; furthermore, there are several constraints on the scaling-up process. Molecular farming has successfully been used for large-scale production in a short time and has been approved for production of several pharmaceutical proteins and vaccines [9][10][11][12][13]. Agroinfiltration is the commonly used technique in molecular farming that involves Agrobacterium tumefaciens-mediated transformation and production of recombinant proteins in plant cells for several days [14][15][16]. This transient protein expression is accomplished when A. tumefaciens transfers a specific part of its Ti (tumor-inducing) plasmid for integration into the genome of infected plant cells [17]. The flexibility and high speed of plant-based expression systems make them especially suitable for vaccine development in the management of outbreaks, epidemics, and pandemics by offering a prompt response though rapid production of subunit vaccines [18]. For example, molecular farming-based vaccines against influenza and SARS CoV-2 viruses have been approved for human use in Canada [19][20][21].
In addition to producing purified pharmaceutical proteins, molecular farming can also be used to produce edible transgenic plants to potentially serve as oral, edible vaccines. Edible vaccines are as safe as conventional virus vaccines and they do not require extensive processing, expensive purification, cold-chain transport, or sterile delivery by trained professionals [9]. The idea behind oral vaccines is that the antigens will be protected from gastric acid and enzymes by the plant cell walls, and can subsequently be delivered to the mucosal surfaces of the gastrointestinal tract to evoke an efficient mucosal immune response [22].
Virus-like particles (VLPs) are structures formed by the self-assembly of viral structural proteins. The VLPs mimic virus morphology but lack infectious genomic material [23], and their small size and the repetitive epitope patterns on their surfaces trigger a strong immune response [24]. These qualities make VLPs strong candidates for vaccine development, and it has been shown that a plant-based quadrivalent VLP influenza vaccine provides better protection than egg-based vaccines in humans [25]. A wide range of plant-based VLPs (including hepatitis B virus, influenza virus, human-and bovine-papillomavirus, bluetongue virus, norovirus, and cowpea mosaic virus) have been shown to elicit protective immune responses in animal models, and have been evaluated as vaccine candidates or for novel delivery systems [26]. In addition, VLPs for several flaviviruses, including TBEV, have been produced by solitary expression of the structural proteins in various hosts and evaluated as potential vaccine candidates [27].
The objective of this study was to explore transient expression of TBEV structural proteins in plants for developing plant-based edible vaccines in which plant leaves expressing the TBEV VLPs would be consumed directly. In addition, we evaluated whether the TBEV antibodies could be vertically transmitted from immunized female mice to offspring and provide protection against TBEV infection.

Recombinant Binary Vectors
A pEAQ-CME vector encoding TBEV structural proteins was constructed using TBEV C-prM-E sequence (accession number DQ401140.3). To increase expression, the TBEV genomic sequence was codon-optimized for the model plant Nicotiana tabacum (a close relative of one of our experimental plants), chemically synthesized, and cloned into a plant expression vector pEAQ-HT using NruI and XhoI restriction sites. A Kozak consensus sequence GCCACC was incorporated upstream of the TBEV start codon. A pEAQ-GFP Vaccines 2022, 10, 1667 3 of 11 vector was constructed using pEAQ vector [28] and a pJL3:p19 vector with p19 gene of tomato bushy stunt virus (TBSV) [29] was kindly provided by Mike Boehm.

Transformation
The recombinant vectors were transformed into competent A. tumefaciens strain LBA4404 using standard procedures and one minute freezing in liquid nitrogen [30]. The transformants were selected on LB plates with antibiotics (kanamycin 50 µg/mL, rifampicin 25 µg/mL and streptomycin 50 µg/mL). Plates were incubated at 28 • C for 2-3 days or until colonies appeared. Single colonies of A. tumefaciens transformed with a binary vector were separately inoculated into 3 mL LB media with antibiotics (kanamycin 50 µg/mL, rifampicin 25 µg/mL, streptomycin 50 µg/mL) and incubated at 28 • C for 2 days at 220 rpm. The culture was pelleted by centrifugation and re-suspended in 100 µL ddH2O. The resuspension was boiled for 5 min and centrifuged at 5000× g for 1 min. The supernatant was used as PCR template to confirm transformants containing pEAQ-CME vector. Colony PCR was performed using the forward primer 5 -TCTCTACTTCTGCTTGACGAGG-3 , the reverse primer 5 -AAGCTTGATATCGAATTCCCGG-3 and Phusion high-fidelity DNA polymerase (New England BioLabs). The resulting PCR products were analyzed by electrophoresis.

Plant Growth
The seeds of 5 different species were germinated and grown in a growth room with a 12 h light/dark cycle. Plants were agroinfiltrated at specified weeks post germination to maximize transient protein expression: a close relative of commercial tobacco (Nicotiana benthamiana), 6 weeks; New Zealand spinach (Tetragonia tetragonioides), 9 weeks; lettuce (Lactuca sativa), 5 weeks; Swiss chard (Beta vulgaris), 7 weeks; spinach (Spinacia oleracea), 5 weeks. Plants were kept in sealed cultivation chambers with artificial light following agroinfiltration. The N. benthamiana used in this study is classified as a low or non-converter of alkaloids [30].

Syringe Infiltration
A. tumefaciens transformed with pEAQ-CME, pEAQ-GFP or pJL3:p19 were grown separately to an OD 600 of 0.8-1.0 in LB medium supplemented with kanamycin 50 µg/mL, rifampicin 25 µg/mL and streptomycin 50 µg/mL at 28 • C with shaking (225 rpm). The bacterial cultures were mixed using 35 mL of A. tumefaciens/pEAQ-CME or A. tumefaciens/pEAQ-GFP and 15 mL of A. tumefaciens/pJL3:p19 and pelleted by centrifugation at 5000× g for 10 min. The pellet was resuspended in freshly made MMA solution infiltration buffer (10 mM 2-N-morpholinoethanesulfonic acid pH 5.6, 10 mM MgCl2, and 100 µM acetosyringone) and stir-incubated at room temperature in the dark for 2-4 h. Mixed cultures of A. tumefaciens were infiltrated into the abaxial surface of each leaf with 1 mL needleless syringe applying gentle pressure to minimize leaf damage.

GFP Expression
Plants were moved to a dark room and exposed to a hand-held UV lamp emitting 366 nm long wave UV light to visualize GFP expression in leaves. Photos were taken without a flash to visualize green fluorescence 7 days post infiltration (dpi).

Immunoblotting
Plant leaves were harvested 7 dpi and ground in liquid nitrogen to a fine powder using a pestle and mortar. Protein extraction was performed in RIPA buffer prior to boiling in 1xLDS sample buffer (Invitrogen). Proteins were separated by SDS-PAGE using precast 4-12% Bis-Tris gels (Invitrogen) and MES running buffer (Invitrogen). The proteins were transferred to nitrocellulose membranes using iBlot 2 Gel Transfer Device (Invitrogen) for detection with an in-house rabbit anti-TBEV E polyclonal antibody

Transmission Electron Microscopy
The samples were negative stained using 3 µl of the sample applied on glow-discharged carbon-coated and formvar-stabilized 400 mesh copper grids (Ted Pella) and incubated for approximately 30s. Excess of sample was blotted off and the grid was washed with MilliQ water prior to negative staining using 2% uranyl acetate. TEM imaging was done using HT7700 (Hitachi High-technologies) transmission electron microscope operated at 100 kV and equipped with a 2kx2k Veleta CCD camera (Olympus Soft Imaging System).

Animal Experiments
BALB/cAnNRj 5-6 weeks old mice were housed at the Norwegian Institute of Public Health, Oslo, Norway. Mice were originally purchased from JANVIER LABS (Le Genest-Saint-Isle, France) and acclimatized for one week. They were housed in plastic cages in a room with a 12 h light/dark cycle and controlled humidity (55 ± 5%) and temperature (20-24 • C). All mice were fed ad libitum with a standard maintenance diet from Special Diets Services (Witham, UK).
To test the oral toxicity of different plant species, a total of 70 female mice were housed in groups of five and fed diets supplemented with different plants for 48 h. N. benthamiana, L. sativa, and B. vulgaris were given as 25 g, 50 g, and 100 g supplements, while S. oleracea was given as 25 g and 50 g supplements, and T. tetragonioides was given as a 25 g supplement. The control mice were fed the standard maintenance diet. The mice were weighed on day 0 and on day 28 (at termination of the trial), and they were euthanized by cervical dislocation. All organs were examined for toxicity by gross pathological examination.
To test immunization, a total of 40 female and 5 male mice were housed in groups of five. The mice were immunized by feeding with 25 g of N. benthamiana leaves expressing TBEV VLPs (n = 10) or with 0.5 mL subcutaneous injections of Ticovac TM (Pfizer, New York, NY, USA) (n = 10) at weeks 0, 2, and 4. To control for the effect of the plant, mice (n = 10) were fed 25 g PBS-infiltrated N. benthamiana leaves whereas control mice (n = 15) were fed the standard maintenance diet. Blood samples were taken from each mouse before each immunization. After week 5, half the mice (n = 5) from each treatment and control group were challenged with 10 6 PFU of TBEV strain Hochosterwitz in 100 µL cell culture media by subcutaneous injection above the right foreleg. Control mice for TBEV challenge were injected with 100 µL of cell culture media. The mice were monitored for pathological symptoms and survival time. The remaining female mice (n=5) from each treatment and control group were mated with untreated males, and offspring were infected with the same batch of TBEV that had been used to infect the maternal mice. The mice were monitored and euthanized by cervical dislocation and all organs were examined for toxicity by gross pathological examination.

Transient Protein Expression in N. benthamiana after Agroinfiltration
To study transient protein expression by agroinfiltration in N. benthamiana (tobacco) and T. tetragonioides (New Zealand spinach), cultures of A. tumefaciens transformed with pEAQ-GFP and pJL3:p19 were mixed and infiltrated. The pJL3:p19 was co-infiltrated to express p19 protein, a suppressor of gene silencing, to enhance expression of GFP by pEAQ-GFP vector. N. benthamiana leaves emitted strong green fluorescence 7 dpi reflecting successful GFP expression, whereas T. tetragonioides leaves showed almost no fluorescence ( Figure 1B,C). We repeated the syringe agroinfiltration three times, but T. tetragonioides failed to express GFP. In fact, the bacterial suspension could not spread through T. tetragonioides in the same way that it did in N. benthamiana, and damage to the leaf structure was observed when we increased the infiltration pressure. reflecting successful GFP expression, whereas T. tetragonioides leaves showed almost no fluorescence ( Figure 1B,C). We repeated the syringe agroinfiltration three times, but T. tetragonioides failed to express GFP. In fact, the bacterial suspension could not spread through T. tetragonioides in the same way that it did in N. benthamiana, and damage to the leaf structure was observed when we increased the infiltration pressure.

Successful Expression and Processing of TBEV Structural Proteins in N. benthamiana
Successful expression of flavivirus structural proteins requires post-translational cleavage by virus and host proteases. To investigate if TBEV structural proteins can be successfully expressed in tobacco, cultures of A. tumefaciens transformed with pEAQ-CME and pJL3:p19 were infiltrated into N. benthamiana. A band of ~50 kDa corresponding to TBEV E protein was visualized by both polyclonal and monoclonal antibodies against flavivirus E protein (Figure 2A,B). The transmission electron microscopy of pEAQ-CME-infiltrated N. benthamiana leaves showed circular particles of ~50 nm diameter ( Figure 2C) which may have been formed by post-translational processing of C-prM-E polyprotein and self-assembled into VLPs.

Successful Expression and Processing of TBEV Structural Proteins in N. benthamiana
Successful expression of flavivirus structural proteins requires post-translational cleavage by virus and host proteases. To investigate if TBEV structural proteins can be successfully expressed in tobacco, cultures of A. tumefaciens transformed with pEAQ-CME and pJL3:p19 were infiltrated into N. benthamiana. A band of~50 kDa corresponding to TBEV E protein was visualized by both polyclonal and monoclonal antibodies against flavivirus E protein (Figure 2A,B). The transmission electron microscopy of pEAQ-CMEinfiltrated N. benthamiana leaves showed circular particles of~50 nm diameter ( Figure 2C) which may have been formed by post-translational processing of C-prM-E polyprotein and self-assembled into VLPs.
reflecting successful GFP expression, whereas T. tetragonioides leaves showed almost no fluorescence ( Figure 1B,C). We repeated the syringe agroinfiltration three times, but T. tetragonioides failed to express GFP. In fact, the bacterial suspension could not spread through T. tetragonioides in the same way that it did in N. benthamiana, and damage to the leaf structure was observed when we increased the infiltration pressure.

Successful Expression and Processing of TBEV Structural Proteins in N. benthamiana
Successful expression of flavivirus structural proteins requires post-translational cleavage by virus and host proteases. To investigate if TBEV structural proteins can be successfully expressed in tobacco, cultures of A. tumefaciens transformed with pEAQ-CME and pJL3:p19 were infiltrated into N. benthamiana. A band of ~50 kDa corresponding to TBEV E protein was visualized by both polyclonal and monoclonal antibodies against flavivirus E protein (Figure 2A,B). The transmission electron microscopy of pEAQ-CME-infiltrated N. benthamiana leaves showed circular particles of ~50 nm diameter ( Figure 2C) which may have been formed by post-translational processing of C-prM-E polyprotein and self-assembled into VLPs.

Orally Administrated N. benthamiana Was Tolerated in Mice
Although N. benthamiana showed high GFP expression and successful production of TBEV VLPs, it may have limitations as a vector for oral immunization due to the risk of toxicity conferred by alkaloids. To study the toxicity of the various plant species in mice, we fed them a diet supplemented with leaves from five different plant species (N. benthamiana, T. tetragonioides, L. sativa, B. vulgaris, and S. oleracea). The edible plant species (all other than N. benthamiana) were used as controls to test these species for future development of oral vaccines. All plant species were accepted by the mice and eaten in comparable amounts (consumption of N. benthamiana was slightly reduced for the two highest amounts of leaf material). No general effect of these supplements on animal weight was observed except for a slightly lower weight gain for the mice that were fed the diet supplemented with S. oleracea or the higher doses of N. benthamiana (50 g and 100 g) ( Table 1). The acute toxicology study was conducted according to OECD 420 guidelines. The animals in control group 2 gained substantially less weight than the animals in control group 1 and one animal was removed from calculation of the average because it did not show any weight gain after 28 days. Weight gain of all animals in control groups was 1.3-3.0 g. The plant leaves were readily consumed by the mice and moisture loss in the plants was not taken into the calculation, since the observed loss was <0.36%. All animals appeared healthy throughout the study, and they showed healthy organs in the gross pathological examination by the veterinarian.

Immunization with Commercial TBEV Vaccine Protected the Offspring
We were interested in testing the immunogenicity of our edible vaccine candidate and comparing it to the commercial TBEV vaccine, Ticovac. We immunized the animals by feeding them fresh N. benthamiana leaves expressing TBEV VLPs or with Ticovac injection. After 5 weeks, 50% of the immunized and control animals were infected with TBEV. All animals immunized with edible vaccine candidates and in control groups died 6-8 days after TBEV infection, while the mice that received Ticovac survived the challenge ( Table 2). The gross pathological examination showed that the animals fed with N. benthamiana leaves had comparatively smaller organs than the other animals. All the mice that died after TBEV challenge showed massive bleeding in the stomach and upper part of the duodenum. We also wanted to test if the mice immunized with the developed vaccine candidate and/or Ticovac could transmit sufficient antibodies to offspring to protect them against TBEV infection. For this experiment, the remaining 50% of the immunized and control animals were mated with untreated males. Their offspring were challenged with TBEV on day 21 post-birth and monitored for pathological symptoms and survival time. The offspring mice that had been vertically immunized (that is, their mothers had been vaccinated with Ticovac) remained healthy after TBEV challenge, while offspring from the other groups died 6-8 days post TBEV infection ( Table 2).

Discussion
In this study, we used transient expression of TBEV C-prM-E in N. benthamiana (a tobacco relative) as a first step in an attempt to develop an edible TBE vaccine. We evaluated the oral toxicity of various plant species and immunogenicity of the edible vaccine candidate in mice. Flavivirus VLPs are commonly produced by expressing a polyprotein comprising C-prM-E or prM-E that is post-translationally cleaved into individual proteins that selfassemble to produce mature VLPs [27]. In this study, we used a pEAQ-CME vector to express C, prM, and E of TBEV as a single polyprotein. The pEAQ vectors are well characterized for VLP production in plants [31,32]. Flavivirus prM protein stabilizes the E protein, and molecular interaction between prM and E is important for flavivirus VLP production [33]. Flavivirus E protein has an indispensable role in cellular binding and membrane fusion between virus and host cell [34,35]. E protein is one of the most potent flavivirus antigens because it contains several epitopes actively recognized by the host immune system and it is a primary target of neutralizing antibodies [36]. Analysis of pEAQ-CME-derived expression of TBEV structural proteins in N. benthamiana by immunoblotting showed a band of~50 kDa when stained with flavivirus anti-E antibodies. This observation indicates that N. benthamiana can provide a suitable environment, similar to mammalian cells, for post-translational processing of the polyprotein to produce correctly cleaved E protein. Similar observations showing ER insertion and post-translational processing of HBsAg in plants have been reported previously [37]. In addition, atomic resolution cryo-electron microscopy has demonstrated that the VLPs of cowpea mosaic virus (CPMV) produced in N. benthamiana are identical to native virus particles [38].
The choice of viral vectors used to express foreign proteins depends on the plant host and the size of the gene to be expressed so that concomitant protein folding is not disturbed. Binary vectors derived from plants viruses, such as tobacco mosaic virus (TMV), tobacco rattle virus (TRV), CPMV, and potato virus X (PVX) have been used to express recombinant foreign proteins in plants [31,39], and transient expression of vaccine antigens in tobacco after Agrobacterium-mediated transformation with TMV-and CPMV-based viral vectors has previously been shown [40,41]. In this study, we used a CPMV-based vector to express TBEV structural proteins in N. benthamiana by agroinfiltration. In addition, we used a second vector encoding the gene-silencing suppressor p19 from TBSV because p19 has been demonstrated to have enhanced transient expression in plants [42]. We used sequences of TBEV structural genes that had been codon-optimized for expression in N. tabacum to achieve higher expression levels of recombinant proteins in N. benthamiana [41][42][43]. Indeed, we achieved high protein expressions at 7 dpi, which is consistent with previous studies [29,41].
An ideal candidate plant for edible vaccine development would have high expression of recombinant proteins and non-toxic traits. In this study, we used N. benthamiana and T. tetragonioides (New Zealand spinach) for recombinant protein expression, and the former showed much stronger expression than the latter. T. tetragonioides was used as a putative edible transgenic plant system expressing a VLP vaccine candidate that could be consumed for immunization. The two factors that might have hindered agroinfiltration and subsequent transient protein expression in T. tetragonioides are its leaf morphology (in terms of thickness, cell composition, or density) and the fact that the expression system was optimized for N. tabacum. The other plant we investigated, N. benthamiana, is a versatile model for replication of plant viruses and expression of recombinant protein using virus-derived expression vectors [44]. However, this non-edible tobacco relative is not particularly attractive as an edible vaccine platform due to the toxic alkaloids in its leaves, primarily the addictive and toxic nicotine, but also smaller amounts of nornicotine (a suspected carcinogen [45]), anatabine, and anabasine [46]. The N. benthamiana used in the present study is classified as a low or non-converter of alkaloids. In the toxicity experiment, we did not observe any visible gross pathological changes in the animals except a slightly lower weight gain in animals that were fed with higher doses of N. benthamiana (50 g and 100 g). Nevertheless, we chose to proceed with low doses (25 g) in the immunization experiment. The mice were fed with fresh N. benthamiana leaves because curing leaves is known to elevate the proportion of nornicotine to nicotine in the leaves [46]. Despite these measures, the immunization experiments showed that the mice fed with N. benthamiana had smaller organs than the other mice, and it has been previously shown that tobacco can cause significant reduction in body and organ weights in mice and rats [47,48].
Despite their potential advantages, edible vaccines face the problem of dose quantification and quality control in the raw plants without purification [49]. In the immunization experiment in which mice were fed with N. benthamiana leaves expressing TBEV structural proteins, we did not observe any noticeable immune response. The lack of adequate immune responses after oral immunization could be due to several factors, including problems with stability in the gastrointestinal tract, dosage, protein folding or modifications, a need for repeated exposure, and lack of adjuvant. It has been reported that feeding mice with raw plants requires an adjuvant or priming with purified VLPs to trigger an optimal immune response [37]. The immunogenicity of the current vaccine may be improved by using an appropriate adjuvant [50], which, however, requires additional studies.
Flaviviruses such as JEV, TBEV, and WNV mainly cause encephalitis [51] while other flaviviruses like YFV, DENV, Omsk haemorrhagic fever virus, and Kyasanur Forest disease virus can cause haemorrhage [52,53]. We observed massive bleeding in the stomachs and upper parts of the duodenum in all the mice that died after infection with TBEV strain Hochosterwitz. These rare but important observations were supported by the clinical findings of TBEV and JEV infections in Russia and India [54,55].
Maternal immunization, which induces virus-specific antibodies that are subsequently dispensed to the offspring through placenta or breastfeeding, remains an effective public health strategy against several virus infections. In this study, female mice vaccinated with Ticovac and their offspring showed a 100% survival rate after TBEV challenge, and none of the infected animals developed any clinical symptoms (this example of maternal immunization in mice corroborates a recently published work based on another liveattenuated flavivirus vaccine [56]). The observations of this study are based on an edible TBEV vaccine candidate and immunization in mice, and extrapolations to other viruses and humans should not be made at this time. In conclusion, we have shown that TBEV structural proteins can be expressed in N. benthamiana and self-assembled to form~50 nm particles similar to TBEV virions. An oral toxicity experiment showed that N. benthamiana is tolerated in mice. However, immunization with the edible vaccine candidate could not protect the mice against TBEV challenge. The commercially-available TBE vaccine Ticovac provided complete protection to both female mice and their offspring.